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Item Efeitos da suplementação com creatina e cafeína sobre a força de fratura óssea em ratos submetidos a exercício de saltos verticais(Revista da Educação Física / UEM, 2012-01) Oliveira, Tânia Toledo; Gomes, Gilton de Jesus; Silva, Karina Ana da; Natali, Antônio José; Costa, Neuza Maria Brunoro; Franco, Frederico Souzalima CaldoncelliEste estudo investigou se a suplementação com creatina e cafeína, isoladamente ou combinadas, interfere na força de fratura óssea em ratos jovens exercitados. Os ratos foram divididos aleatoriamente em oito grupos: sedentários placebo, exercitado placebo, sedentário creatina, exercitado creatina, sedentário cafeína, exercitado cafeína, sedentário creatina + cafeína e treinado creatina + cafeína. Os grupos suplementados receberam creatina (carga: 0,430g/kg de peso corporal, por sete dias; e manutenção: 0,143g/kg, por 35 dias), cafeína (10mg/100g de peso corporal, por 35 dias) ou creatina+cafeína. Os grupos exercitados executaram saltos verticais na água (4x10 saltos com 1 minuto de intervalo entre séries, 5 dias/sem) por seis semanas. A ingestão de cafeína reduziu a espessura, o peso e a força de fratura do fêmur dos ratos, independentemente do exercício. A cafeína e a creatina+cafeína aumentaram a excreção urinária de cálcio. O exercício de saltos elevou a força de fratura, independentemente da suplementação, mas não alterou o peso e as dimensões do fêmur dos animais.Item Avaliação da concentração de proteínas e da atividade de α-galactosidases nos cotilédones e no eixo embrionário de sementes de Dalbergia nigra durante a germinação(Acta Amazonica, 2011) Carrijo, Lanna Clicia; Borges, Eduardo Euclydes de Lima e; Rezende, Sebastião Tavares de; Pontes, Cláudia Aparecida; Silva, Aderlan Gomes da; Lopes, Mariana RochaEste trabalho teve como objetivos a quantificação de proteínas e da atividade da enzima α-galactosidase, no eixo embrionário e nos cotilédones, de sementes de Dalbergia nigra (jacarandá-da-bahia) durante a germinação. As sementes foram colocadas para embeber em água por sete dias, sendo retiradas amostras para a avaliação bioquímica e cinética da enzima. A atividade da enzima α-galactosidase aumenta com a embebição das sementes nos dois compartimentos, embora não esteja presente no eixo embrionário de sementes secas. A diferença na atividade da enzima entre os cotilédones e o eixo embrionário foi significativa. O pH 5,5 foi o de máxima atividade para as enzimas de ambos os compartimentos. A temperatura que mais estimulou a atividade da enzima nos cotilédones foi 50 ºC e de 50 a 60 ºC no eixo embrionário. A atividade da α-galactosidase foi inibida por β-mercaptoetanol e cobre, em ambos os compartimentos, enquanto a lactose e o cloreto de sódio estimularam a atividade tanto nos cotilédones como no eixo embrionário. Os valores de KM para enzimas do eixo embrionário e dos cotilédones foram de 0,239 e 0,228 mM, respectivamente.Item RBCS1 expression in coffee: Coffea orthologs, Coffea arabica homeologs, and expression variability between genotypes and under drought stress(BMC Plant Biology, 2011) Ramos, Humberto J. O.; Marraccini, Pierre; Freire, Luciana P.; Alves, Gabriel S. C.; Vinecky, Felipe; Elbelt, Sonia; Montagnon, Christophe; Vieira, Luiz G. E.; Leroy, Thierry; Pot, David; Silva, Vânia A.; Rodrigues, Gustavo C.; Andrade, Alan C.; Vieira, Natalia G.In higher plants, the inhibition of photosynthetic capacity under drought is attributable to stomatal and non-stomatal (i.e., photochemical and biochemical) effects. In particular, a disruption of photosynthetic metabolism and Rubisco regulation can be observed. Several studies reported reduced expression of the RBCS genes, which encode the Rubisco small subunit, under water stress. Expression of the RBCS1 gene was analysed in the allopolyploid context of C. arabica, which originates from a natural cross between the C. canephora and C. eugenioides species. Our study revealed the existence of two homeologous RBCS1 genes in C. arabica: one carried by the C. canephora sub-genome (called CaCc) and the other carried by the C. eugenioides sub-genome (called CaCe). Using specific primer pairs for each homeolog, expression studies revealed that CaCe was expressed in C. eugenioides and C. arabica but was undetectable in C. canephora. On the other hand, CaCc was expressed in C. canephora but almost completely silenced in non-introgressed ("pure") genotypes of C. arabica. However, enhanced CaCc expression was observed in most C. arabica cultivars with introgressed C. canephora genome. In addition, total RBCS1 expression was higher for C. arabica cultivars that had recently introgressed C. canephora genome than for "pure" cultivars. For both species, water stress led to an important decrease in the abundance of RBCS1 transcripts. This was observed for plants grown in either greenhouse or field conditions under severe or moderate drought. However, this reduction of RBCS1 gene expression was not accompanied by a decrease in the corresponding protein in the leaves of C. canephora subjected to water withdrawal. In that case, the amount of RBCS1 was even higher under drought than under unstressed (irrigated) conditions, which suggests great stability of RBCS1 under adverse water conditions. On the other hand, for C. arabica, high nocturnal expression of RBCS1 could also explain the accumulation of the RBCS1 protein under water stress. Altogether, the results presented here suggest that the content of RBCS was not responsible for the loss of photosynthetic capacity that is commonly observed in water-stressed coffee plants. We showed that the CaCe homeolog was expressed in C. eugenioides and non-introgressed ("pure") genotypes of C. arabica but that it was undetectable in C. canephora. On the other hand, the CaCc homeolog was expressed in C. canephora but highly repressed in C. arabica. Expression of the CaCc homeolog was enhanced in C. arabica cultivars that experienced recent introgression with C. canephora. For both C. canephora and C. arabica species, total RBCS1 gene expression was highly reduced with WS. Unexpectedly, the accumulation of RBCS1 protein was observed in the leaves of C. canephora under WS, possibly coming from nocturnal RBCS1 expression. These results suggest that the increase in the amount of RBCS1 protein could contribute to the antioxidative function of photorespiration in water-stressed coffee plants.Item Identification and expression levels of pig miRNAs in skeletal muscle(Livestock Science, 2013-06) Verardo, L. L.; Nascimento, C. S.; Silva, F. F.; Gasparino, E.; Toriyama, E.; Barbosa, A. R.; Costa, K. A.; Lopes, P. S.; Guimarães, S. E. F.; Périssé, I. V.MicroRNAs are a class of naturally occurring non-coding RNAs. Typically they are $22 nucleotides long and suppress translation of their targets genes. Several laboratories have attempted to identify miRNAs from pig muscle and the bioinformatics strategies using ESTs have proved to be successful for this aim. In this study we report an in silico identification of ncRNA in pig EST libraries focusing on novel pig miRNAs and further investigated the differential expression of pigs miRNAs (known and novel) by quantitative real-time PCR during preand postnatal stage from Commercial and local breed Piau pigs skeletal muscle tissue. We identified two miRNAs not yet described in pigs: hsa-miR-1207-5p and hsa-miR-665. Besides, we found 288 target genes for hsa-miR-1207-5p and 214 for hsa-miR-665; from them, four are muscle specific genes. Through expression analyses, differences were found between pre- and postnatal stages and genetics groups. The findings of miRNAs and their muscle-specific targets in pigs will be helpful for understanding the function and processing of this RNA class in the future. Besides, the miRNAs differentially expressed between Commercial and Piau breeds suggest that they can be used to uncover phenotypic differences across different genetic groups.Item Concentração de proteína solúvel por bradford revela diferenças no metabolismo de plantas de ora-pro-nobis em diferentes doses de nitrogênio(Revista Brasileira de Agropecuária Sustentável, 2013-07) Guimarães, Dimitrius Santiago P. Simões Fróes; Souza, Maria Regina de Miranda; Hirano, Rafael T.; Pereira, Paulo Roberto Gomes; Baracat-Pereira, Maria CristinaO ora-pro-nobis (Pereskia aculeata Mill.) é um alimento rico em proteínas, muito utilizado no meio rural e com importância crescente na indústria de alimentos e farmacológica. O objetivo deste trabalho foi determinar a concentração de proteínas pelos métodos de Bradford e Kjeldahl, em folhas de plantas de ora-pro-nobis submetidas a diferentes doses de adubação nitrogenada, comparando estes métodos. As folhas das plantas de ora-pro-nobis adubadas com diferentes doses de N (0, 50, 100, 200 e 400 kg de N/ha) foram coletadas aos 423 dias após o plantio (DAP). Para o método de Bradford, as folhas foram trituradas com nitrogênio líquido e maceradas em Tris-HCl 50 mM, pH 7,0, o homogenato centrifugado e a proteína solúvel determinada no sobrenadante. Para avaliar o perfil proteico, as amostras dos diferentes tratamentos foram separadas por SDS-Tricina-PAGE 14%. O método de Kjeldahl tradicional foi realizado usando-se o fator de correção 6,25. Os resultados por ambos os métodos indicaram que houve alterações nas concentrações e composição de proteínas presentes em função da disponibilidade de N no solo. A proteína total por Kjeldahl aumentou até a dose de 100 kg de N/ha, e a proteína solúvel por Bradford aumentou nas doses de N entre 50 e 200 kg/ha. Pelo SDS-Tricina-PAGE, verificou-se aumento da intensidade das bandas consonante com o método de Bradford. Estes resultados sugerem que a avaliação de proteínas solúveis pelo método de Bradford permite detectar diferenças no metabolismo das plantas de ora-pro-nobis, expressando informações biológicas relevantes para estudos fisiológicos e nutricionais.Item The involvement of calcium carriers and of the vacuole in the glucose-induced calcium signaling and activation of the plasma membrane H + - ATPase in Saccharomyces cerevisiae cells(Cell Calcium, 2012-01) Bouillet, L. E. M.; Fietto, L. G.; Cardoso, A. S.; Perovano, E.; Pereira, R. R.; Ribeiro, E. M. C.; Trópia, M. J. M.; Tisi, R.; Martegani, E.; Castro, I. M.; Brandão, R. L.Previous work from our laboratories demonstrated that the sugar-induced activation of plasma membrane H+-ATPase in Saccharomyces cerevisiae is dependent on calcium metabolism with the contribution of calcium influx from external medium. Our results demonstrate that a glucose-induced calcium (GIC) transporter, a new and still unidentified calcium carrier, sensitive to nifedipine and gadolinium and activated by glucose addition, seems to be partially involved in the glucose-induced activation of the plasma membrane H+-ATPase. On the other hand, the importance of calcium carriers that can release calcium from internal stores was analyzed in glucose-induced calcium signaling and activation of plasma membrane H+-ATPase, in experimental conditions presenting very low external calcium concentrations. Therefore the aim was also to investigate how the vacuole, through the participation of both Ca2+-ATPase Pmc1 and the TRP homologue calcium channel Yvc1 (respectively, encoded by the genes PMC1 and YVC1) contributes to control the intracellular calcium availability and the plasma membrane H+-ATPase activation in response to glucose. In strains presenting a single deletion in YVC1 gene or a double deletion in YVC1 and PMC1 genes, both glucose-induced calcium signaling and activation of the H+-ATPase are nearly abolished. These results suggest that Yvc1 calcium channel is an important component of this signal transduction pathway activated in response to glucose addition. We also found that by a still undefined mechanism Yvc1 activation seems to correlate with the changes in the intracellular level of IP3. Taken together, these data demonstrate that glucose addition to yeast cells exposed to low external calcium concentrations affects calcium uptake and the activity of the vacuolar calcium channel Yvc1, contributing to the occurrence of calcium signaling connected to plasma membrane H+-ATPase activation.Item Ecto-nucleotidase activities of promastigotes from Leishmania (Viannia) braziliensis relates to parasite infectivity and disease clinical outcome(PLOS Neglected Tropical Diseases, 2012-10-11) Fietto, Juliana L. R.; Leite, Pauline M.; Gomes, Rodrigo S.; Figueiredo, Amanda B.; Serafim, Tiago D.; Tafuri, Wagner L.; Souza, Carolina C. de; Moura, Sandra A. L.; Melo, Maria N.; Ribeiro-Dias, Fátima; Oliveira, Milton A. P.; Rabello, Ana; Afonso, Luı́s C. C.Leishmania (Viannia) braziliensis has been associated with a broad range of clinical manifestations ranging from a simple cutaneous ulcer to destructive mucosal lesions. Factors leading to this diversity of clinical presentations are not clear, but parasite factors have lately been recognized as important in determining disease progression. Given the fact that the activity of ecto-nucleotidases correlates with parasitism and the development of infection, we evaluated the activity of these enzymes in promastigotes from 23 L. braziliensis isolates as a possible parasite-related factor that could influence the clinical outcome of the disease.Our results show that the isolates differ in their ability to hydrolyze adenine nucleotides. Furthermore, we observed a positive correlation between the time for peak of lesion development in C57BL/6J mice and enzymatic activity and clinical manifestation of the isolate. In addition, we found that L. (V.) braziliensis isolates obtained from mucosal lesions hydrolyze higher amounts of adenine nucleotides than isolates obtained from skin lesions. One isolate with high (PPS6m) and another with low (SSF) ecto-nucleotidase activity were chosen for further studies. Mice inoculated with PPS6m show delayed lesion development and present larger parasite loads than animals inoculated with the SSF isolate. In addition, PPS6m modulates the host immune response by inhibiting dendritic cell activation and NO production by activated J774 macrophages. Finally, we observed that the amastigote forms from PPS6m and SSF isolates present low enzymatic activity that does not interfere with NO production and parasite survival in macrophages.Our data suggest that ecto-nucleotidases present on the promastigote forms of the parasite may interfere with the establishment of the immune response with consequent impaired ability to control parasite dissemination and this may be an important factor in determining the clinical outcome of leishmaniasis.Item Evaluation of the effects of Quercetin and Kaempherol on the surface of MT-2 cells visualized by atomic force microscopy(Journal of Virological Methods, 2011-04-11) Almeida, Marcia R.; Reis, Jordana Grazziela A. Coelho dos; Gomes, Orlando Ab; Bortolini, Dener E.; Martins, Marina L.; Martins, Camila S.; Carvalho, Luciana D.; Souza, Jaqueline G.; Vilela, Jose Mario C.; Andrade, Margareth S.; Stancioli, Edel Figueiredo BarbosaThis study investigated the anti-viral effects of the polyphenolic compounds Quercetin and Kaempherol on the release of HTLV-1 from the surface of MT-2 cells. Atomic force microscopy (AFM) was used to scan the surface of the MT-2 cells. MT-2 cells were fixed with 100% methanol on round glass lamina or cleaved mica and dried under UV light and laminar flow. The images were captured on a Multimode equipment monitored by a NanoScope IIId controller from Veeco Instruments Inc operated in tapping mode and equipped with phase-imaging hardware. The images demonstrated viral budding structures 131 ± 57 nm in size, indicating profuse viral budding. Interestingly, cell-free viruses and budding structures visualized on the surface of cells were less common when MT-2 was incubated with Quercetin, and no particles were seen on the surface of cells incubated with Kaempherol. In summary, these data indicate that HTLV-1 is budding constantly from the MT-2 cell surface and that polyphenolic compounds were able to reduce this viral release. Biological samples were analyzed with crude cell preparations just after cultivation in the presence of Quercetin and Kaempherol, showing that the AFM technique is a rapid and powerful tool for analysis of antiviral activity of new biological compounds.Item High-yield secretion of multiple client proteins in Aspergillus(Enzyme and Microbial Technology, 2012-07-15) Gonçalves, Thiago A.; Segato, Fernando; Damásio, André R. L.; Lucas, Rosymar C. de; Squina, Fabio M.; Decker, Stephen R.; Prade, Rolf A.Production of pure and high-yield client proteins is an important technology that addresses the need for industrial applications of enzymes as well as scientific experiments in protein chemistry and crystallization. Fungi are utilized in industrial protein production because of their ability to secrete large quantities of proteins. In this study, we engineered a high-expression-secretion vector, pEXPYR that directs proteins towards the extracellular medium in two Aspergillii host strains, examine the effect of maltose-induced over-expression and protein secretion as well as time and pH-dependent protein stability in the medium. We describe five client proteins representing a core set of hemicellulose degrading enzymes that accumulated up to 50–100 mg/L of protein. Using a recyclable genetic marker that allows serial insertion of multiple genes, simultaneous hyper-secretion of three client proteins in a single host strain was accomplished.Item Diversity of soil-dwelling Trichoderma in Colombia and their potential as biocontrol agents against the phytopathogenic fungus Sclerotinia sclerotiorum (Lib.) de Bary(Journal of General Plant Pathology, 2012-11-24) Smith, Alexander; Beltrán, Camilo A.; Kusunoki, Manabu; Cotes, Alba M.; Motohashi, Keiichi; Kondo, Takumasa; Deguchi, MichihitoTwenty-one isolates of Trichoderma spp. were collected from eight states in Colombia and characterized based on the 5′ end of the translation elongation factor-1α (EF1-α1) gene and RNA polymerase II gene encoding the second largest protein subunit (RPB2) by using mixed primers. Seven species of soil-dwelling Trichoderma were found: T. atroviride, T. koningiopsis, T. asperellum, T. spirale, T. harzianum, T. brevicompactum and T. longibrachiatum. Species identifications based on the EF1-α1 gene were consistent with those obtained from the RPB2 gene. Phylogenetic analyses with high bootstrap values supported the validity of the identification of all isolates. These results suggest that using the combination of the genes EF1-α1 and RPB2 is highly reliable for molecular characterization of Trichoderma species. Trichoderma asperellum Th034, T. atroviride Th002 and T. harzianum Th203 prevented germination of more than 70 % of sclerotia of Sclerotinia sclerotiorum in bioassay tests and are promising biological control agents. No relationship between mycelium growth rate and parasitism level was found.